This project is devoted to detailed analysis of pyridoxal enzyme systems using advanced instrumental analytical techniques. The objective is three-fold: 1) to develop and improve the system of computerized instrumentation for spectroscopy, 2) to investigate the usefulness of low temperature luminescence and photo excited EPR spectroscopy in studies of pyridoxal systems, and 3) to investigate the details of the catalytic mechanism of the pyridoxal-copper enzyme, hog kidney diamine oxidase. The low temperature fluorescence and phosphorescence of pyridoxal compounds has not yet been exploited as an analytical tool for studies of pyridoxal enzymes. Several features of these measurements suggest their utility. Of even more interest, however, is the electron paramagnetic resonance signal associated with the photoexcited triplets of these and related compounds. Using a variety of spectroscopic tools such as stopped-flow, difference spectroscopy, and polarization of fluorescence spectroscopy along with the power of the computer instrument systems to correlate and manipulate a variety of data, the sequence of reaction events and the role of copper in diamine oxidase will be investigated. The studies of diamine oxidase are directed toward development of a model of the kinetic mechanism of this enzyme. The first studies have revealed a complex anaerobic half reaction with multiple enzyme intermediate species. These investigations will also pursue the reoxidation half reaction mechanism.